PROJECT SUMMARY. Disruptions to sexual desire are a leading cause of patient noncompliance, greatly in- creasing risk to mental and physical wellbeing. Furthermore, reduced sexual desire is highly comorbid with neurological, cardiovascular, and metabolic diseases, with up to 20% of the US population reporting long-term, personally-distressing impairments in sexual motivation. This represents a significant public health concern as sexual dysfunction contributes to the etiology of mood disorders via decreased quality of life, reduced self- esteem, and increased strain on interpersonal relationships. There is a critical need for further investigation into the neural substrates of sexual motivation to facilitate development of efficacious treatments for sexual de- sire dysfunctions. Lateral hypothalamic neurons that produce the neuropeptide hypocretin (Hcrt, also known as orexin) are strong candidates to mediate sexual drive. Hcrt neurons promote arousal and reward-seeking be- havior, and this proposal?s preliminary data demonstrates that Hcrt neuronal activity is highly associated with the expression of sexual motivation. However, the circuit mechanisms underlying Hcrt regulation of sexual mo- tivation remain unclear. The medial preoptic area of the hypothalamus (mPOA) is essential for reproductive behavior across vertebrates and receives dense innervation by Hcrt neurons, making it an ideal candidate to mediate Hcrt actions on sexual motivation. Therefore, the central hypothesis of the present proposal is that mPOA-projecting Hcrt neurons (HcrtmPOA) promote sexual motivation in males and females. This hypothesis will be tested in three aims: Aim 1 will define endogenous HcrtmPOA neuronal activity in sociosexual contexts. HcrtmPOA calcium activity will be recorded using fiber photometry as male and female animals are exposed to a variety of social and nonsocial stimuli and during mating. The relationship between HcrtmPOA neuronal activity and specific stimuli, sexual behaviors, and motivational transitions will be assessed. Aim 2 will determine whether HcrtmPOA activity is necessary for the natural expression of sexual motivation. HcrtmPOA activity will be chemogenetically suppressed and the impact on sexual motivation, anxiety-like behavior, reward-seeking be- havior, and physiological arousal (via simultaneous EEG/EMG recording) will be assessed. Aim 3 will deter- mine whether stimulation of HcrtmPOA neuronal activity is sufficient to rescue sexual motivation in hormone- deficient animals or to escalate sexual motivation in hormone-competent animals. HcrtmPOA neurons will be optogenetically stimulated and the impact on behavior assessed as in Aim 2. HcrtmPOA activity is expected to be associated with expression of sexual motivation, and to specifically regulate sexual motivation without impacts on other behavioral metrics. Successful completion of the proposed studies will provide outstanding technical training, imparting skills in fiber photometry, chemogenetics, optogenetics, and EEG/EMG recording. This work will be completed within the de Lecea and Shah labs at Stanford University, which will provide an exemplary training environment and comprehensive development of conceptual and professional skills.